Our objectives are to determine the molecular structure of one or more transfer RNA molecules and to determine the functional roles of various facets of that structure. The modified bases, the characteristic loops (D, psi, variable anticodon loops) and the 3-d folding all need to be related to function if we are to understand possible abberations of genetic degenerative, infectious or nutritional diseases which might effect tRNA function. Our methods are to isolate, purify, and study tRNA by solution chemistry and crystallography. We use specific chemical and enzymic modification to probe the surface of the molecule in solution and to add heavy atoms for x-ray crystallographic study. The bulk of the effort is consumed in tRNA purification and in development of chemical techniques for selective modification of bases in tRNA. We will also prepare extremely high purity alkaline phosphatase, CCA-ligase and certain aa- tRNA synthetases for this RNA research. We have developed procedures for labeling surface cytidylic acids on tRNA with iodine and have crystallized the labeled tRNA. Mercury, lead and gold derivatives of tRNA have been crystallized. tRNA has been prepared with mercury attached to the amino acid on the tRNA. These materials are needed for x-ray structure analysis of tRNA. In the coming year we will scale up the production and crystallization of these tRNA derivatives and collect x-ray diffraction data from them. We will also test the suitability for x-ray analysis of a mercury derivative of aminoacyl tRNA which we developed in the past year. We have developed the techniques of "Ion filtration" for rapid isolation of enzymes and have prepared several enzymes bound to glass beads for use in RNA structure studies.